Overexpression And Activity Analysis Of Antrodia Camphorata Immunomodulatory Protein

Antrodia camphorata immunomodulatory protein(ACA)is an important fungal immunomodulatory protein isolated from Antrodia camphorata.Wild Antrodia cinnamomea only parasitize in the decaying trunks of over 100 years old cinnamomum camphora,one Class II protected tree in Taiwan.There was little data about the pharmacological and clinical research on ACA because of the rarity,preciousness and nonexportation of Antrodia camphorata.To reduce the costs of ACA expression,3 engineering bacteria,p ET-32-a-ACA/BL21,p ET-32-a-ACAb/BL21 and p GEX-4t-2-ACA/BL21 were constructed,then p ET-32-a-ACA/BL21 was screened out as the most suitable engineered bacteria after inducing the expression of rACA,protein expression conditions and medium formulations were optimized based on the engineered bacteria subsequently.Finally,the activity analysis of ACA were performed after purification,which laid the foundation for the industrial production of ACA.The main results of this study were as follows:1.Construction and screening of engineering bacteria.To explore the effects of the codon bias and the vectors on protein expression,wild type ACA gene and gene with E.coli codon bias were synthesized through OE-PCR using DNAworks 3.2.4 to design and optimize primers,and named as ACAb and ACA respectively.Then,the correct ACAb gene was inserted into expression vector p ET-32-a,ACA gene was inserted into p ET-32-a and p GEX-4t-2 respectively,and the recombinant vectors were transformed into E.coli and named as p ET-32-a-ACAb/BL21,p ET-32a-ACA/BL21and p GEX-4t-2-ACA/BL21 respectively after sequencing,and the expression conditions of the three engineered bacteria were optimized subsequently.The results showed that p ET-32-a-ACA/BL21 was more suitable for the expression of rACA,the optimal expression condition was found to be inducing p ET-32-a-ACA/BL21 with 0.6mmol/L IPTG,for 7 h at 25?.2.Optimization of fermentation conditions for complete medium.Four commonly complete media LB,TB,SOB and 2YT were used to induce the expression of the selected engineering bacteria p ET-32-a-ACA/BL21,under the same conditions,the expression effect of SOB was better than the other three,and with the yield of137.37?g/m L.In order to increase the expression of rACA,SOB medium was optimized using response surface methodology.The Plackett-Burnman experiment,the steepest climbing experiment,the central combination design(CCD)and response surface analysis were carried out subsequently.The results showed that the optimal fermentation conditions were at yeast powder 8.92?g/m L in SOB medium,initial p H7.0,inoculation volume 1%,liquid volume 10 m L/50 m L,IPTG 0.42 mmol/L,shake speed of 200 rpm,at 25?for 7 h.Under these conditions,187.12?g/m L rACA was obtained after fermentation,which was 36.22%higher than the initial SOB(137.37?g/m L).3.Optimization of cheap medium formula.SOB culture is expensive and not suitable for industrial production.It is imperative to find a low-cost culture medium.Corn steep liquor and alcohol fermentation waste liquor were used as raw materials,both the one-factor-at-a-time(OFAT)method and the response surface methodology(RSM)were used to optimize the medium formula.The OFAT method indicated that the optimum conditions for rACA expression were that Na Cl,KCl and Mg Cl₂other than trace elements,with corn steep liquor 8 g/L,and ethanol waste liquor 500 m L/L.Under this condition,the expression level of rACA reached 160.075?g/m L.The optimum medium using RSM were found to be corn steep liquor 14.11 g/L,ethanol waste 209.16 m L/L,Na Cl 0.5 g/L,KCl 2.5 mmol/L,Mg Cl₂ 10 mmol/L,and p H 7.0.When fermented the engineered strain p ET-32-a-ACA/BL21 in optimized medium,181.424?g/m L rACA was obtained after inducing with 0.4 mmol/L IPTG,with a speed of 200 rpm for 7 h at 25°C,with the initial filling volume 10 m L/50 m L,which was 11.77%higher than the OFAT and close to the outputs from optimized SOB.4.rACA activity detection.To evaluate the activity of rACA,Mouse macrophages RAW264.7 was treated with purified recombinant His-rACA(recombinant ACA,rACA),then cell proliferation,phagocytosis,changes in cell morphology,and NO,tumor necrosis factor-?(TNF-?),Interleukin-1?(IL-1?)and other cytokines were measured.At the same time,Hela cells,Caco-2 cells and mouse myeloma cells SP2/0 were treated with rACA to detect the effects of rACA on the cell viability of cancer cells,The results showed that purified His-rACA could not only significantly promote the proliferation of mouse macrophages,improved the phagocytic activity of cells,changed the cell morphology,and significantly induced macrophages to secrete NO,TNF-?and IL-1?,but also inhibit the proliferation of Hela cells and Caco-2 cells to varying degrees.Intristingly,rACA had no inhibitory effect on mouse myeloma cell SP2/0.The high-efficiency and soluble expression of rACA and the activity similar to natural ACA enable it to replace natural ACA as a food additive or immunomodulator in the pharmaceutical industry,and could even be used in drug research.